Pichia production host engineering by systemic host changes

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Unfolded protein response biosensors for recombinant protein expression

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Promoter and process engineering for recombinant protein production in Pichia pastoris towards simple, fast and methanol-free cultivation regimes and high product titers

Protein production in Pichia pastoris often applies methanol-induced gene promoters such as PAOX1 to drive the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties independent of methanol such as the PGTH1 promoter which we reported recently [1]. In order to further increase its potential, we investigated its regulation in more detail by screening of promoter variants harbouring deletions and mutations. Thereby we could identify the main regulatory region and important transcription factor binding sites of PGTH1. We also created a PGTH1 variant, called PG1-3, with greatly enhanced induction properties compared to the wild type promoter. Model based process engineering could successfully be implemented for PG1-3 to outperform the PAOX1-driven production in a simple feed regime, and to establish a speed fermentation with high titers after only two days total fermentation time. [1] Prielhofer, R.; Maurer, M.; Klein, J.; Wenger, J.; Kiziak, C.; Gasser, B.; Mattanovich, D., Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris. Microb Cell Fact 2013, 12 (1), 5

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Utility of nutritional evaluation for the clinical risk assessment of postoperative complications. Does oncology need the NRS 2002 scale?

Introduction. Malnutrition accompanies many cancers, especially those of the gastrointestinal tract, and significantly raises the risk of postoperative complications in cancer surgery. In Poland, hospitals are legally obliged to evaluate the nutritional status of their patients; one of the assessment tools used for this purpose is the NRS 2002 scale. Aim. The primary objective of the study is to analyze the utility of the NRS 2002 scale in the risk assessment of posto­perative complications in gastrointestinal cancers. In addition, the authors propose to determine whether the legal requirement to conduct nutritional assessments among hospitalized patients is complied with in clinical practice and to evaluate the risk of malnutrition in the study group. Materials and methods. A detailed assessment was conducted on 226 patients who underwent surgery for upper (95 patients) and lower (131 patients) GI tract cancers in 2015. The risk of complications was analyzed based on the nutritional risk score (NRS 2002) and the levels of albumin and total proteins in the serum before surgery. Compliance with the obligation to carry out nutritional assessments was evaluated on breast and GI cancer patients treated with surgery at the Institute of Oncology in Warsaw in two successive years. Results. An NRS 2002 score of ≥ 3 was shown to predict postoperative complications for both upper GI tract (p < 0.001) and colorectal cancers (p < 0.001). In upper GI cancers, complications were also more frequently observed at lower albumin (p = 0.018) and total protein (p = 0.025) levels in the serum. Conclusion. The analysis shows that the NRS 2002 scale is useful in predicting the risk of postoperative complications in the treatment of upper and lower GI tract cancers

Przydatność klinicznej oceny stanu odżywienia do oceny ryzyka powikłań pooperacyjnych Czy skala NRS 2002 jest w onkologii potrzebna?

Wstęp. Niedożywienie to stan często towarzyszący nowotworom, szczególnie przewodu pokarmowego. W przypadku zabiegu operacyjnego zwiększa ono ryzyko powikłań pooperacyjnych. W Polsce każdy szpital jest zobowiązany do oceny stanu odżywienia swoich pacjentów. Jedną ze skal umożliwiających taką prostą ocenę jest NRS 2002. Cel. Celem pracy jest analiza przydatności skali NRS 2002 w ocenie ryzyka powikłań pooperacyjnych w leczeniu nowotworów przewodu pokarmowego. Cele drugorzędne to sprawdzenie, czy obowiązek oceny stanu odżywienia chorych hospitalizowanych jest realizowany w praktyce klinicznej oraz jakie jest ryzyko niedożywienia w analizowanej grupie. Materiał i metody. Ocenie szczegółowej poddano 226 chorych operowanych w 2015 roku z powodu nowotworów górnego (95 chorych) i dolnego odcinka przewodu pokarmowego (131 chorych). Ryzyko powikłań analizowano w zależności od oceny ryzyka żywieniowego (NRS 2002), przedoperacyjnego poziomu albumin oraz białka całkowi­tego w surowicy krwi. Ocenie częstości wypełniania skali poddano w dwóch kolejnych latach chorych operowanych z powodu nowotworów piersi i przewodu pokarmowego w Centrum Onkologii w Warszawie. Wyniki. Wynik oceny NRS 2002 ≥ 3 był czynnikiem prognostycznym powikłań pooperacyjnych zarówno dla no­wotworów GOPP (p &lt; 0,001), jak i dla nowotworów okrężnicy (p &lt; 0,001). Dla nowotworów gopp potwierdzono częstsze występowanie powikłań również w przypadku obniżenia poziomu albumin w surowicy (p = 0,018) i białka całkowitego (p = 0,025). Wnioski. Przeprowadzona analiza potwierdza, że skala NRS 2002 jest przydatnym narzędziem w przewidywaniu ryzyka powikłań pooperacyjnych w leczeniu nowotworów górnego i dolnego odcinka przewodu pokarmowego

An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles

Nitrilase from Rhodococcus rhodochrous ATCC 33278 hydrolyses both aliphatic and aromatic nitriles. Replacing Tyr-142 in the wild-type enzyme with the aromatic amino acid phenylalanine did not alter specificity for either substrate. However, the mutants containing non-polar aliphatic amino acids (alanine, valine and leucine) at position 142 were specific only for aromatic substrates such as benzonitrile, m-tolunitrile and 2-cyanopyridine, and not for aliphatic substrates. These results suggest that the hydrolysis of substrates probably involves the conjugated π-electron system of the aromatic ring of substrate or Tyr-142 as an electron acceptor. Moreover, the mutants containing charged amino acids such as aspartate, glutamate, arginine and asparagine at position 142 displayed no activity towards any nitrile, possibly owing to the disruption of hydrophobic interactions with substrates. Thus aromaticity of substrate or amino acid at position 142 in R. rhodochrous nitrilase is required for enzyme activity

Characterisation of the substrate specificity of the nitrile hydrolyzing system of the acidotolerant black yeast Exophiala oligosperma R1

The `black yeast' Exophiala oligosperma R1 can utilise various organic nitriles under acidic conditions as nitrogen sources. The induction of a phenylacetonitrile converting activity was optimised by growing the strain in the presence of different nitriles and /or complex or inorganic nitrogen sources. The highest nitrile hydrolysing activity was observed with cells grown with 2-cyanopyridine and NaNO3. The cells metabolised the inducer and grew with 2-cyanopyridine as sole source of nitrogen. Cell extracts converted various (substituted) benzonitriles and phenylacetonitriles. They usually converted the isomers carrying a substituent in the meta-position with higher relative activities than the corresponding para- or ortho-substituted isomers. Aliphatic substrates such as acrylonitrile and 2-hydroxy-3-butenenitrile were also hydrolysed. The highest specific activity was detected with 4-cyanopyridine. Most nitriles were almost exclusively converted to the corresponding acids and no or only low amounts of the corresponding amides were formed. The cells hydrolysed amides only with extremely low activities. It was therefore concluded that the cells harboured a nitrilase activity. The specific activities of whole cells and cell extracts were compared for different nitriles and evidence obtained for limitation in the substrate-uptake by whole cells. The conversion of 2-hydroxy-3-butenenitrile to 2-hydroxy-3-butenoic acid at pH 4 demonstrated the unique ability of cells of E. oligosperma R1 to hydrolyse aliphatic α-hydroxynitriles under acidic conditions. The organism could grow with phenylacetonitrile as sole source of carbon, energy and nitrogen. The degradation of phenylacetonitrile presumably proceeds via phenylacetic acid, 2-hydroxyphenylacetic acid, 2,5-dihydroxyphenylacetic acid (homogentisate), maleylacetoacetate and fumarylacetoacetate